Human MSCs attenuated LPS-induced lung inflammation and injury in immunocompetent mice. In response to the pro-inflammatory environment in the LPS exposed lung, hMSCs upregulated expression of a number of genes. Some of these genes encode secreted factors with known anti-inflammatory properties. One of the most highly upregulated genes was TNFAIP6 that encodes TSG-6. Knockdown of TSG-6 mRNA expression in hMSCs abrogated most of their anti-inflammatory effects. In addition, intra-pulmonary delivery of rhTSG-6 reduced LPS-induced inflammation in the lung. These results suggest that hMSCs in the lung upregulated expression of TNFAIP6 and the secreted TSG-6 decreased neutrophil accumulation and lung injury.
Previous studies have demonstrated the ability of murine MSCs to attenuate inflammation and lung injury in rodent models of ALI [19, 24, 25]. Recently, the ability of human MSCs to restore alveolar epithelial fluid transport and lung fluid balance was demonstrated in a study employing an ex vivo perfused human lung preparation injured by E. coli endotoxin . These studies have ignited much interest in hMSCs as cellular therapy for ALI. However, little is known about the mechanisms by which MSCs modulate lung inflammation and repair. The anti-inflammatory effects of mouse MSCs in the lung have been associated with MSC secretion of interleukin 1 receptor antagonist (IL1RN)  and TGF-β1 . It is not clear how closely mMSCs resemble hMSCs. Certainly these two populations of cells share many features; however, there is some evidence that mMSCs and hMSCs differ in their response to inflammatory cytokines. For example, Ren et al. showed in vitro that inflammatory cytokines induce the expression of indoleamine 2,3-dioxygenase (IDO), but not inducible nitric oxide synthase (iNOS), in human MSCs, whereas in mouse MSCs the opposite is true . The goal of our study was to investigate the mechanism(s) by which human MSCs modulate inflammation in the lung.
For these studies we delivered human MSCs to immunocompetent mice after intrapulmonary delivery of LPS, a well-characterized mouse model of direct lung injury that recapitulates many of the features of human ALI including patchy intra-alveolar neutrophil infiltrates and changes in epithelial permeability. A similar cross-species strategy was used to elucidate the mechanism(s) by which hMSCs improve heart function after myocardial infarction . In that study, hMSCs were delivered to immunodeficient mice where they provided numerous endogenous markers for the cells and caused no obvious cross-species artifacts.
Our results demonstrated that hMSCs improved lung histology and reduced pulmonary edema in immunocompetent mice (Figure 1) similar to what has been reported for syngeneic mMSCs in this model [19, 24]. The improvement in lung injury was associated with a decrease in proinflammatory cytokines and chemokines, including some (IL-1α, IL-1β, MIP-1α and MIP-2) that function as chemoattractants and activators of neutrophils (Figures 2d and 3). Consistent with these results, hMSC treated lungs had significantly fewer neutrophils, the major mediator of lung injury in this model (Figure 2b). TNF-α levels in BAL fluid were not changed with hMSC treatment and although there was a trend toward increased IL-10 in BAL fluid the levels of IL-10 were very low (Supplemental Table S1 in Additional file 2). Administration of HLFs had no beneficial effects (Figure 2 and Supplemental Figure S2 in Additional file 3). These results suggest that hMSCs protected the lung primarily by preventing recruitment and/or activation of neutrophils. In a study similar to ours, mouse MSCs given four hours after LPS attenuated lung injury and reduced the levels of TNF-α and MIP-2 in BAL fluid. However, neutrophil influx or activation was not ameliorated . The differences in the MSC effects on neutrophil influx may be explained by intrinsic differences in MSCs from humans versus mice, but could also be explained by differences in experimental conditions between the studies, including strain of mice used, dose of LPS and method of intra-pulmonary delivery.
The ability of human MSCs to attenuate lung inflammation and injury in an immunocompetent mouse suggests that the anti-inflammatory action of hMSCs in the lung is non-major histocompatibility complex (MHC)-restricted and further, that hMSCs are to some degree immune privileged due to their immune modulatory effects, as others have suggested . These results support the use of allogeneic hMSCs in patients.
Our finding that hMSCs attenuated lung inflammation when administered intravenously (Figure 4) was not surprising since most of the injected cells are initially trapped in the lung . However, this is the first report that MSCs delivered intraperitoneally attenuate inflammation in the lung (Figure 4b, c). The hMSCs did not travel to the lung after IP injection as indicated by the absence of human GAPDH mRNA in the lung one hour (Figure 4a) or six hours (data not shown) after injection. The fate of the cells is currently under investigation. The IP route of delivery was not as effective as OA and IV routes in reducing LPS-induced lung injury. For these experiments, we used a dose of hMSCs (5 × 105) that we determined to be optimal for OA delivery. It is possible that increasing the number of hMSCs administered IP would provide better protection. Alternatively, our results may suggest that the factor or factors responsible for hMSC suppression of inflammation can function systemically, whereas factors that enhance lung repair may work best when delivered locally. Regardless, these results strongly suggest that the beneficial properties of hMSCs are predominantly mediated through secretion of factors that act in paracrine fashion.
The upregulation of TNFAIP6 by hMSCs in response to the inflammatory milieu of the LPS-exposed lung (Table 1) was of special interest because TSG-6 has previously been shown to play a role in the beneficial effects of hMSCs . TSG-6 is a 35 kDa, secreted protein produced by many cell types in response to TNF-α and IL-1β [20, 21]. hMSCs in the LPS-exposed lung upregulated their TSG-6 mRNA expression more than 900-fold (Figure 5a). TSG-6 is composed mainly of contiguous Link and CUB modules that interact with a broad spectrum of glycosaminoglycans and proteins, including HA, chondroitin 4-sulphate, dermatan sulfate, heparan sulfate, heparin, inter-α-inhibitor, versican, aggrecan, thrombospondin-1, and PTX3 . The anti-inflammatory properties of TSG-6 are well documented and the mechanisms are beginning to be elucidated. TSG-6 inhibits the inflammatory network of proteases by increasing the inhibitory activity of inter-α-inhibitor and bikunin . TSG-6 also specifically binds and sequesters hyaluronan fragments and has been shown to be a potent inhibitor of neutrophil activation and migration, as well as tissue remodeling , through up-regulation of Cox-2 and prostaglandin D2 expression [28, 29]. The precise mechanism(s) by which TSG-6 protects the lung from acute inflammation remains to be determined.
Knockdown of TSG-6 mRNA expression in hMSCs did not completely abrogate their anti-inflammatory effects (Figure 6) suggesting that additional factors expressed by hMSCs play a role in protecting the lung from LPS-induced inflammation and injury. The hMSCs used in our studies expressed high levels of KGF in the lung (Figure 5b). Lee et al. showed in an isolated, perfused human lung model challenged with endotoxin, that secretion of KGF was essential for the beneficial effect of hMSCs on alveolar epithelial fluid transport . In another study, Aguilar et al.  showed that MSCs transfected with a tetracycline-inducible KGF construct partially protected mice from bleomycin-induced pulmonary fibrosis. The mechanisms underlying KGF's protective effect in the lung are not completely understood. KGF has plieotropic effects on alveolar epithelial cells including; mitogenic acitivity, increased surfactant production , inhibition of apoptosis , and increased transcription and/or translation of the major sodium and chloride transport proteins . However, KGF is not known to have direct anti-inflammatory activity. We postulate that TSG-6 and KGF secreted by hMSCs cooperate to attenuate lung injury by suppressing inflammation and promoting lung repair respectively. MSCs secrete other bioactive molecules such as IL1RN (Figure 5c), HGF, EGF, TGF-β1, sTNFR1, Ang1 and STC-1 that may also contribute to their immunomodulatory functions and repair of injured lung [19, 26, 34–38]. Additional experiments are required to determine the relative contribution of each of these factors to the beneficial effects of hMSCs in the lung.
It is tempting to speculate that strategies to enhance hMSC expression of TSG-6 and other beneficial factors such as KGF may improve their lung protective properties. One such strategy is to culture hMSCs as 3D aggregates or as spheroids [39–41]. Recently it was shown that hMSCs cultured in hanging drops formed spheroids and expressed significantly higher levels of TSG-6 and STC-1 compared to hMSCs cultured as monolayers. Moreover, hMSCs from dissociated spheroids were more effective at suppressing inflammation in a mouse model of peritonitis than were hMSCs cultured as adherent monolayers . Using a similar strategy we showed that culture of hMSCs in spheroids increased their expression of TSG-6, STC-1 and KGF (unpublished data). Our finding that hMSCs delivered IP have beneficial effects in the lung suggests the possibility of transplantation of the intact spheroids. We are currently comparing the therapeutic potential of traditionally cultured hMSCs to that of spheroid hMSCs in our animal model of ALI.